An inability or reduced ability to have children can cause great personal distress and has a high attendant social cost, particularly in terms of the cost of medical intervention. A large proportion of couples fall into this category. In the USA, for example, it is said that some 10–15% of couples of reproductive age are unable to have children, whereas in the United Kingdom this is 14%. In 1995 it was calculated that 5.1 million women had impaired fertility in the USA alone, with this figure projected to increase to 5.9 million by the year 2020 (56). In the US, the cost of a pregnancy conceived by IVF varies between US$66,000 for the first cycle to US$114,000 by the sixth cycle (60).
In the context of this specification an infertility condition is to be understood to relate not only the capacity to conceive but also the miscarriage, spontaneous abortion or other pregnancy-related conditions, such as pre-eclampsia, and includes sub-fertility.
Recent studies have revealed that a major proportion of interfile couples are childless because of a higher than normal rate of early embryonic loss (70% miscarriage v 21% miscarriage in fertile controls: 57), rather than an inability to conceive. These findings have initiated a search for reasons for the increased rate of early embryonic loss in infertile couples, as well as potential therapies to avert such losses.
In the last 20 years or so some hope has been held out to infertile couple with the development of in vitro fertilisation (IVF) techniques. These IVF techniques generally take the form of stimulating the female to ovulate, contacting collected ova with sperm in vitro and introducing fertilised ova into the uterus. Multiple variations of this general process also exist. Despite considerable research and technical advances in the IVF field the rate of successful pregnancy following IVF treatment is still quite low and is in the order of 15 to 25% per cycle.
Undertaking an IVF program often causes great anguish, especially when there is no resultant successful pregnancy. It is presently believed that the poor success rate in IVF treatment is due to an extraordinarily high rate of early embryonic loss (58, 59), possibly related to the patient's impaired reproductive state or the IVF process itself.
The low efficacy of IVF, together with its high cost and the associated psychological trauma from repeated treatment failures makes it desirable that alternative approaches to the problem of infertility are sought. Current methods of increasing pregnancy rates during IVF treatment include placing multiple embryos (2–5) into the uterine cavity, but this is not always effective since uterine receptivity is believed to be at fault at least as commonly as embryonic viability. Furthermore, the ensuing high rates of multiple pregnancy are associated with an increased material risk of pre-eclampsia, haemorrhage and operative delivery, and fetal risks including pre-term delivery with the attendant possibility of physical and mental handicap.
Similarly, early pregnancy loss is a major constraint in breeding programs for livestock and rare or threatened species. Embryonic mortality during the pre- and per-implantation period is viewed as the major reason for poor pregnancy outcome when assisted reproductive technologies such as artificial insemination are used. Even following natural mating, variability in litter size and in the viability of offspring are additional limitations with serious economic implications.
The reasons for increased rates of early embryonic loss following natural and assisted conception remain unknown. Chromosomal studies on miscarried embryos have confirmed that at least half of these embryos are genetically normal (61). Normal embryos appear to be lost primarily because the environment provided by the material tract during pre-implantation development or at the time of implantation into the endometrium is insufficient to nurture their growth and development. Embryos may lose viability or developmental potential if the material tract milieu comprises inappropriate or insufficient nutrients or peptide growth factors. Moreover, a primary determinant of uterine receptivity is provided by the maternal immune response to the conceptus, which is perceived as foreign or semi-allogeneic due to expression of both maternal and paternal antigens.
Medawar originally hypothesised that maternal immune accommodation of the semi-allogeneic conceptus may be facilitated by immunological tolerance to paternal transplantation antigens (major histocompatibility [MHC] antigens)(70). This hypothesis lost favour when it was found that pregnancy does not permanently alter the capacity of mice to reject paternal skin grafts (5, 46). However, the concept of transient hyporesponsive to paternal MHC antigens (46) is now receiving renewed attention, as a recent study by Tafuri et al (31) has provided clear evidence to show that during murine pregnancy, T-lymphocytes reactive with paternal class 1 MHC become ‘anergic’, or unable to recognise antigen due to internalisation of T-cell receptors. This anergic state conferred ‘tolerance’ to paternal MHC antigen-expressing tumor cells, and was functionally operative from as early as implantation (day 4 of pregnancy) and lasted until shortly after parturition when lymphocytes regained their reactivity. The data support the hypothesia that a permissive maternal immune response to other antigens expressed on the embryo, or the fetal-placental unit (hereinafter referred to as the conceptus) may similarly be due to induction of a tolerant immune response specific to those antigens.
Just precisely what is responsible for inducing this tolerance of paternal MHC antigens and other conceptus antigens has heretofore been unclear. Additionally the nature of the tolerance was unclear.
The term tolerance in the context of this invention is taken to mean inhibition of the potentially destructive cell-mediated immune response against conceptus antigens, and/or inhibition of synthesis of conceptus antigen-reactive immunoglobulin of complement-fixing isotypes (for example the ‘Th1’ compartment of the immune response). This tolerance may or may not be associated with induction of synthesis of non-destructive, conceptus antigen-reactive immunoglobulin of the non-complement-fixing isotypes and subclasses (for example the ‘Th2’ compartment of the immune response). The term tolerance should be taken to encompass T cell energy and other permanent or transient form of hypo-responsiveness or suppression of the maternal Th1 compartment.
Tafuri et al (31) have shown that paternal antigen-specific tolerance is active by the onset of blastocyst implantation on day 4 of pregnancy in mice. The pre-implantation embryo is a poor antigenic stimulus since it usually comprises fewer than 100 cells and is enveloped by a protective coat (zona pellucidé) until just before implantation. Semen however is richly endowed with paternal antigens present on and within sperm, somatic cells and the seminal plasma itself, and comprises an effective priming inoculum for many paternal antigens (5) known to be shared by the conceptus. Up until now seminal plasma has been conventionally thought to function primarily as a transport and survival medium for spermatozoa traversing the female reproductive tract (21). The recent studies described by the inventors in this specification have highlighted a hitherto unappreciated role for this fluid in interacting with maternal cells to induce a cascade of cellular and molecular events which ultimately lead to maternal immune tolerance to paternal antigens present in semen and shared by the conceptus, thereby abrogating immune rejection during implantation.
Ejaculation during coitus provokes a leukocyte infiltrate as the site of semen deposition termed the ‘leukocytic cell reaction’ in a variety of mammalian species, including man (1). In mice, the cascade of cellular and molecular changes initiated by the introduction of semen into the uterus, in many respects, resembles a classic inflammatory response. Within hours after mating, a striking influx and activation of macrophages, neutrophils, and eosinophils occurs in the endometrial stroma (2–4), in association with upregulated expression of major histocompatibility complex (MHC) class II and CD86 antigens by endometrial dendritic cells, followed by enlargement of draining lymph nodes (5,6). This inflammatory response is transient and fully dissipates by the time of embryo implantation on day 4 of pregnancy (2–4), when leukocytes persisting in the endometrium are predominantly macrophages with an immunosuppressive phenotype (7).
The temporal changes in trafficking and phenotypic behavior of endometrial leukocytes during the period between mating and implantation are likely to be orchestrated principally by cytokines emanating from steroid hormone regulated epithelial cells lining the endometrial surface and comprising the endometrial glands (8). Of particular importance are granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-(IL)-6, the synthesis of which is upregulated at least 20-fold and 200-fold respectively in estrogen primed epithelial cells following induction by specific proteinaceous factors in seminal plasma (8.9) known to be derived from the seminal vesicle gland (10). Previous studies have implicated the surge in epithelial GM-CSF release as a key mediator in the post-mating inflammatory response since injection of recombinant GM-CSF into the estrous uterus is sufficient to produce cellular changes resembling those seen following natural mating (11). The inventors have found, using GM-CSF deficient mice, that the chemotactic activity of GM-CSF is likely to be compensated or augmented by an array of chemokines, the expression of which is transiently upregulated after mating (12), and cytokines synthesised by activated endometrial macrophage including IL-1 and tumour necrosis factor-α (TNF-α)(4).
The present inventors have investigated the nature of the seminal factor which acts to stimulate GM-CSF release from the uterine epithelium. Previous experiments have shown that the increase in uterine GM-CSF content is neither the result of introduction of GM-CSF contained within the ejaculation, nor a consequence of a neuroendocrine response to cervical stimulation, and is independent both of the presence of sperm in the ejaculation and MHC disparity between the male and female (8). A mechanism involving induction of GM-CSF mRNA synthesis in epithelial cells by proteinaceous factors derived from the seminal vesicle was suggested by experiments showing that seminal vesicle-deficient (SV−) males did not evoke GM-CSF release or a post-mating inflammation-like response in females, and that trypsin-sensitive, high molecular weight material extracted from the seminal vesicle could upregulate GM-CSF release from uterine epithelial cells in vivo (10).
It has, however, not been clear from previously published work that this inflammatory response is related to the induction of tolerance by the mother to the conceptus, or alternatively whether the inflammatory response has a role in enhancing the immune system to combat the influx of foreign matter such as potential pathogenic bacteria is not clear. Nor is there any indication as to what the trigger for the induction of tolerance is or indeed that tolerance is mediated by semen.
One known relevant prior art document is U.S. Pat. No. 5,395,825 by Feinberg. This specification discloses a finding that suggests that elevated TGFβ in the female reproductive tract can facilitate production of fibronectin, a protein hypothesised to assist implantation by promoting adhesion of the embryo to the endometrial surface. The half life TGFβ is only a few minutes and its effect on fibronectin is very short term. Therefore the administration of TGFβ in the above method can only be contemplated to assist implantation is delivered at precisely the time at which the pre-implantation embryo arrives in the uterine cavity. The present invention does not require such temporal precision in TGFβ delivery, nor does it purport that the effect of TGFβ is mediated through fibronectin.